Immunotherapeutic strategies to orient the immune system against tumor cells continue to hold great promise for patients with advanced melanoma. The paucity of conventional treatment options and the complexity and inconsistency of clinical immunotherapy have motivated the development of scientific models to identify ways to improve immunotherapy. Thus far, these models have employed melanoma-specific T cell populations that have been activated and expanded ex vivo for adoptive transfer into lymphodepleted hosts bearing melanoma tumors. Although designed to mimic paradigms of experimental adoptive immunotherapy used in human subjects, the complexity of these models has obfuscated our ability to precisely characterize the in vivo interaction that takes place between adoptively transferred melanoma-specific T cells and melanoma tumors. Our laboratory has developed a simple quantitative animal model of melanoma-induced T cell suppression to demonstrate that in vivo exposure to growing melanoma tumors weakens the ability of T cells to undergo antigen-driven proliferative expansion by heightening their susceptibility to apoptotic cell death. This alteration of T cell responsiveness fundamentally reshapes the entire spectrum of activated T cell homeostasis with one interesting exception: memory T cells appear to be uniquely resistant to this melanoma- induced suppression. In this proposal, we have drawn on our previous experience to introduce new, simplified models of melanoma adoptive T cell immunotherapy to test a central hypothesis: that the efficacy of melanoma adoptive immunotherapy can be optimized with the use of memory T cells. In the first series of experiments, we will test a first subhypothesis: that the in vivo durability of adoptively transferred melanoma-specific T cells is impaired by melanoma-induced upregulation of T cell apoptosis. We will examine the fate of adoptively transferred T cell populations in vivo to characterize how melanoma influences the ability of therapeutic populations of melanoma-specific T cells to persist. In the next series of experiments, we will test a second subhypothesis: that memory T cells are optimal mediators of melanoma adoptive immunotherapy because of an enhanced ability to survive and target melanoma antigens following adoptive transfer. We will compare the in vivo persistence of adoptively transferred melanoma-specific T cells in various stages of maturation (resting T cells, acute effector T cells, unsorted memory T cells, central memory T cells, and effector memory T cells) following adoptive transfer, as well as their abilities to infiltrate melanoma tumors, induc tumoral regression, and promote tumoral immunity. From there, we will test a third subhypothesis: that adoptive immunotherapy is more effective when endogenous melanoma-specific T cell responses are preserved. We will employ highly quantitative assays used in our laboratory to determine how endogenous melanoma-specific T cell responses are affected by adoptive immunotherapy, and will determine if therapeutic efforts to preserve and enhance those endogenous responses promote the efficacy of adoptive immunotherapy. In the fourth and final series of experiments, we will test a fourth subhypothesis: that melanoma-specific memory T cells can be harvested and expanded to quantities that will permit the clinical realization of memory T cell-based adoptive immunotherapy. Here, we will examine human melanoma tumors to verify that the biological advantages of memory T cells can be put to clinical use. This final piece will enable us to begin the next phase of investigation: a clinical rial to introduce a novel and critical form of cancer treatment. In summary, we are proposing a series of experiments with which the biological underpinnings of adoptive T cell immunotherapy may be rationally and critically explored. It is our expectation that this work will allow us to fuly actualize the enormous potential of this desperately needed treatment for veterans afflicted with melanoma. PUBLIC HEALTH RELEVANCE: The incidence of melanoma is increasing faster than that of any other cancer; yet the few treatment options currently available for Veterans with advanced melanoma offer enormous toxicity with minimal-to-no clinical benefit. Experimental efforts to fight melanoma using the patient's own immune system have yielded intriguing but preliminary successes. Our laboratory has developed an experimental way to dissect the biological underpinnings of immunotherapy. We outline a series of experiments that will teach us (1) how melanoma tumors affect the ability of melanoma-specific to persist following adoptive transfer, (2) which immune cells would be most effective in mediating immunotherapy, (3) how current forms of melanoma treatment may synergize with immunotherapy to maximize clinical benefit, and (4) ways to translate these findings into human studies. In short, we will learn ways in which the enormous promise of melanoma immunotherapy may be actualized.